B – CELLS, B CELLS

DEVELOPMENT , ACTIVATION,
B CELLS MARKERS…

SUBMITTED TO: Dr. ARUNA BHATIA

SUBMITTED BY : VANITA
ROLL NO. : 18011010
CLASS: MSC BIOTECHNOLOGY (I)
CONTENTS:
• B CELLS
• B CELL DEVELOPMENT
• B CELL MATURATION
• B CELL CO RECEPTOR
• MARKERS OF B CELLS
WHAT ARE B-CELLS?
• B lymphocyte derived its name from the site of its maturation , in
the bursa of Fabricius in birds.
• Bone marrow is essential for the complete maturation and
differentiation.
• Bone marrow is the major site of maturation in no. of mammalian
species , including humans and mice.
• Mature B cells and definitively distinguished from other cells by their
synthesis and display of membrane –bound immunoglobulin
(antibody) molecules.
• This receptor is highly specific antigen binding site.
• B- cells carries a large number of identical mAbs on the surface, about
1.5 x 105 molecules.
• B-cells express distinctive membrane molecules-B7 ,CD40,BCR,CR1
and CR2.
• Interaction between antigen and the membrane bound antibody on a
mature naive B-cells, interaction T cells and macrophages, induce
activation and differentiation of B cells clones.
• The proliferation and activated B-cell clone comprises two types of
cells:
1. Plasma cells
2. Memory cells
Plasma cells:
• These cells have enormous capacity to produce antibodies of same
specificity as that of antibodies on the original B-cells.
• Rich in cytoplasmic organelles like endoplasmic reticulum.
• Limited life span of few days.
• This process of antibody production constitutes humoral immune
response.
Memory cells:
• These have long life span.
• They express the mAb on their surface of the same antigenic
specificity as that of the original B-cells.
• If the same antigen encounter , these cells immediately recognise and
produce heightened immune response.
B-CELL DEVELOPMENT:
• Development process divided into 3 stages:
1. Generation of mature immunocompetent B-cells(maturation).
2. Activation of mature B-cells when they interact with antigen.
3. Differentiation of activated B cells into plasma and memory B cells.
Process :
The naïve cells is not encountered by antigen , circulate in the blood
and lymph and carried to secondary lymphoid organs.
If the B cells is activated by the antigen specific to its membrane
bound antibody, the cell proliferate ( clonal expansion)and
differentiate into plasma cells and memory cells.
Progressive increase in Change in the isotype of the
the affinity of the antibody produced by the cells
antibody produced. from μ to α, ε, γ.
1. This process is an orderly sequence of Ig gene rearrangement,
progress in the absence of antigen called antigen independent
phase of B-cell development.
2. If the activation and differentiation require antigen , called as
antigen dependent phase of B-cell development .
• Many B-cells are produced in the bone marrow through out life ,
but very few these are marrow . Most of these cells circulate as
naïve B cells which short life span (half lives of less than 3 days
to about 8 weeks) if they fail to encounter to the antigen.
B-CELL MATURATION:
Generation of mature B-cells first occurs in the embryo and continues
throughout life . Major site of B cells:
 Before birth : yolk sac, fetal liver and bone marrow
 After birth: bone marrow
PROGENITOR B CELLS PROLIFERATE IN BONE MARROW:
• Begins as lymphoid stem cells differentiate into the earliest distinctive B
lineage cell- progenitor B cell(pro B cell),express transmembrane
tyrosine phosphate called CD40R(B 20 in mice)
• Pro B cell proliferate and fill the extravascular spaces between the large
sinusoids in the shaft of bone .
• Proliferation and differentiation of pro B cell into precursor B cell(pre B
cell) require micro environment provided by the bone marrow stromal
cells.
• Pro B cell removed and cultured in-vitro, they not progress to more
mature cells unless stromal cells are present.
• Stromal cells plays two important roles:
1. They interact directly with pro B cell.
2. They secrete various cytokines (IL-7) which support the
developmental process.
EARLIEST DEVELOPMENT STAGE:

Pro B cell require direct contact with stromal cells

Interaction is mediated by cell adhesion molecules including VLA-4 on

pro B cell and its ligand VCAM-1 on the stromal cells

After initial contact, a receptor on the pro- B cell called c-Kit interacts
with stromal cell surface molecule known as stem cell factor(SCF).

Activates c-Kit which is tyrosine kinase

Pro B cell begins to divide and differentiate into pre B cell and
expressing a receptor for IL-7

IL-7 secreted by stromal cells

Proliferating cells can detach from stromal cells

At this stage , no longer require direct contact with stromal cell but
require IL-7 for growth and maturation
Ig GENE REARRANGEMENT PRODUCED IMMATURE B- CELLS : B – cell
maturation depends on rearrangement of the immunoglobins DNA in the
lymphoid stem cells.
• First to occur in the pro – B cell stage in a heavy chain Dh to Jh gene
rearrangement then followed to Vh to DhJh rearrangement.
• If the first heavy chain rearrangement in not productive, then VhDhJh
rearrangement continues on the other chromosome.
• Upon completion of heavy chain rearrangement , the cell is classified
as pre B cells.
• Continuous development of pre B cell into immature B cells requires a
productive light chain gene rearrangement.
• Completion of a productive light chain rearrangement , commits the
immature B cells to a particular antigenic specificity determined.
• The bone-marrow phase of B cell development is the production of
an IgM bearing immature B cell.
• Full maturation is signaled by the co- expression of IgM and IgD on
the membrane.
• This progression involves a change in RNA processing of the heavy
chain primary transcript to permit production of mRNA

One encoding the membrane other encoding he membrane

of μ form. Of ꝭ form.
• IgD is a characteristic cell surface marker of the mature naïve B cell
but IgD not essential to either B cell development or antigen
responsiveness.
 PRO CELLS PRE CELLS

(HEAVY CHAIN REARRANGEMENT) (LIGHT GENE REARRANGEMENT)

RAG 1 AND 2 Present Present

TdT Present Absent

(Terminal Deoxyribonucleotidyl (N- nucleotide are not usually found
Transferase) in the VlJl coding joints)

(Catalzes insertion of N-nucleotide

at the Dh-Jh and VhDhJh coding
joints.
THE PRE- CELL RECEPTOR ESSENTIAL FOR B – CELL
DEVELOPMENT:
• In pre B cell , the membrane chain μ in is associated with the surrogate
light chain , a complex consist 2 protein:
1. V like sequence: Vpre-B
2. C Like sequence: λ5
associate non – covalently to form a light chain like structure.
• The membrane bound complex of μ heavy chain and surrogate light chain
appears on the pre B cell associated with Igα/Igβ heterodimer to form the
pre B cell receptor.
• The pre B cell receptor recognizes ligand on the stromal cell membrane,
that transmit signal which prevents Vh to DhJh rearrangement of the other
heavy chain allele leads to allelic exclusion.
• By effective pre-B cell receptor , each pre-B cell undergoes multiplication.
Knockout experiment: identified essential transcription factor:

• Particular transcription factor out by gene distruption which is

important for B cell .
• Eg: the gene encoding the λ5 protein of the receptor was disrupted.

B cell development blocked

Signal through receptor is necessary for pre- B cell to proceed to

immature B cell stage
TRANSCRIPTION FACTORS EFFECT

1. E2A • Do not express RAG1 which unable to make DhJh

rearrangement
• Fail to express λ5 2.

2. EBF(EARLY B CELL FACTOR) • Early stage of commitment to B cell lineage

3. BSAP (B CELL SPECIFIC ACTIVATOR • Found in the promoter region of a number of B cell
PROTEIN) specific genes.
• Role in early stages of B cell development
• Expressed in the central nervous system and absence
results in severe defection in mid brain development.
4. SOX-4 • Affects early stages B cell activation
CELL SURFACE MARKERS IDENTIFY DEVELOPMENT STAGES:
• Progression from progenitor to mature B cell by changing pattern of
surface markers.
• Pro B stage , they do not display the heavy and light chain but express
CD45R( protein tyrosine phosphatase found on leukocytes) and signal
transduction molecule Igα/Igβ.
• Pro B cell also express: CD19( part of B cell co- receptor), CD43
(leukosialin) ,CD24(heat stable antigen –HSA) ,c-Kit.
• When pro B stage into pre B cell that ceases to express c-Kit and CD43,
begins to express CD25,the α chain of the IL-2 receptor.
• The salient feature of pre B cell is pre –B cell receptor.
• After rearrangement of the light chain , surface immunoglobin appears ,
then classified immature B cell, lose pre BCR and CD25.
• Monoclonal antibodies are available that recognize and isolate various
stages of B development by immunohistology and flow cytometer.
B-1 B CELLS ARE A SELF RENEWING B- CELL SUBSET:
• There is a subset of B Cells, called B-1 B cells, arise before B-2 B cells: major
group of B cells in human and mice.
• B-1 B cells compose 5% of the total B cell population.
• Appear during fetal life, express surface IgM but little IgD and marked by
the display of CD5.
• CD5 is not necessary, lack a function CD5 gene still produce B -1 cell.
• During fetal life B-1 cells arise from stem cells in bone marrow.
• B -1 population responds poorly to protein antigens but much better to
carbohydrate.
• Most of the membrane are IgM bearing cells, this undergoes less somatic
hypermutation and class switching than the B-2 set of B cells does.
consequently the antibodies produced by a high proportion of B 1 cells are
of rather low affinity.
SELF REACTIVE B CELLS ARE SELECTED AGAINST IN BONE
MARROW:
• In mouse bone marrow produce about 5× 10000000 cells/day but only
5×1000000 (or about10%) are actually recruited into B cell pool.
• This means 90%of the B cell produced each day die without ever leaving
the bone marrow.
• Some of this loss is attributed able to negative selection and subsequent
elimination (clonal deletion) of immature B cells that express auto
antibodies against self antigen in the bone marrow.
• It is demonstrated experimentally , by treating immature B cells with
antibody against the μ constant region , can cause the cells to die by
apoptosis.
• D.A. Nemazee and K. Burki introduced a transgene encoding heavy and
light chains which results suggest that there is negative selection against
immature B cells expressing autoantibodies on their membranes because
these antibodies react with self antigen.
SELF REACTIVE B CELLS MAY BE RESCUED BY
EDITING OF LIGHT CHAIN GENES:
• Using the transgenic system, described by Nemazee and Burki
showed that negative selection of immature B cells does not always
result in their immediate deletion.
• When some immature B cells bind a self antigen, maturation is
arrested; the cells up-regulate RAG-1 and RAG-2 expression and
further rearrangement of their endogenous light chain DNA.
• Some of these cells succeed in replacing the ĸ light chain of the self
antigen reactive antibody with a λ chain encoded by endogenous λ
chain gene segments.
• As a result, these cells will begin to express an edited mIgM with a
different light chain and specify as not self reactive.
B – CELL ACTIVATION:
• After export of B cells from bone marrow , activation , proliferation
and differentiation occur in the periphery and require antigen.
• Antigen driven activation and clonal selection of naïve B cells lead to
generation of plasma cells and memory cells.
• In the absence of antigen induced activation , naïve B cells in the
periphery have a short life span , die by apoptosis.
THYMUS DEPENDENT AND THYMUS INDEPENDENT:
1. Thymus dependent antigen: direct contact with Th cells
2. Thymus independent antigen: antigen that can activate B cells in
the absence of direct participation of Th cells. Response is weaker
and no memory cells are formed.
 TYPE 1: some bacterial cell wall component , including LPS .
 TYPE 2: polymeric proteins or bacterial cell wall.
Most TI-1 antigens are polyclonal B cell activators(mitogens):they activate B
cell regardless of their antigenic specificity.
 High conc. :stimulate proliferation and antibody secretion as many as one
third of all B cells
 Low conc. :only those B cell specific for epitopes of the antigen will be
activated.
TI-2 : activate B CELLS by cells extensively cross- linking the mIg receptors.
TI-2 antigens differ from TI-1 antigens:
• They are not B-cells mitogens and do not act as polyclonal activators.
• TI-1 activate both mature and immature B cells but TI-2 antigen activate
mature B cell and inactivate immature B cell.
• B cells response to TI-2 antigens doesn’t require direct involvement Th cells,
cytokines are efficient for B cell proliferation and class switching to isotope
other then IgM.
TWO TYPES OF SIGNALS THROUGH CELL CYCLE:
• Naïve B cells are non-dividing cells in the Go stage of cell cycle.
• Activation drives the resting cell into the cell cycle. G1 to S is a critical
restriction point in the cell cycle . if the cell reached S, it completes
cell cycle(G2 to M).
• These events could be grouped into 2 categories:
1. Competence signals: drive the B cell Go to early G1.It is achieved by
two distinct signals : signal 1 and signal 2.
2. Progressive signals: G1 to S then cell division and differentiation.
• These signaling events generated by different pathway( TD , TI) When
multivalent and crosslinks mIg.
• B cell has acquired an effective competence signal in early activation ,
the interaction of cytokines , other ligands ,receptors provide
progression signals.
 TRANSDUCTION OF ACTIVATING SIGNALS
INVOLVES Igα/Igβ HETERODIMER:
• All isotype of mIg have very short cytoplasmic tails.
• Both IgM and IgD on B cells extend into the cytoplasm by only 3 A. acids
(IgM: 14 a.a. , IgG and IgE: 28 a.a.).
• In each case, cytoplasmic tails is too short to be able to generate a signal by
associated with intracellular signaling molecule(tyrosine kinase and G
protein).
• The membrane Ig is associated with disulphide liked heterodimer Igα/Igβ ,
forming B cell receptor (BCR). Which help in ligand binding Ig and signal
transduction respectively.
• The Igα chain has long cytoplasmic tail containing 61 A.A and tail Igβ chain
contains 48 A.A . The cytoplasmic tail containing 18 residue motifs termed
the immunoreceptor tyrosine based activation motifs(ITAM)
• Interaction between cytoplasmic tail and Igα/Igβ transduce the stimulus
produced by cross linking mIg molecule into an effective intracellular signal.
• Signaling from the BCR is mediated by protein tyrosine kinase (PTK) and BCR
itself has no PTK activity like TCR.
• The antigen mediated cross linking of BCR initiate no. of signaling cascades
that ultimately result in cell responses to the cross linking of its surface Ig by
antigen . The cross linking of BCR results many signal transduction pathway
and activation of B cell . many parallel B cell and T cell activation which
include:
1. COMPARTMENTALIZATION OF FUNCTION WITHIN RECEPTOR SUBUNIT:
both B cells and T cells pathway begins with antigen receptor that are
composed of an antigen – binding and a signaling unit.
2. ACTIVATION OF MEMBRANE – ASSOCIATED Src PROTEIN TYROSIN KINASE
ACTIVITY: the receptor associated PTKs (Lck in T cells , Lyn Blk Fyn in B
cells) catalyse phosphorylation . This is essential to form a functional
receptor signaling complex.
3. ASSEMBLY OF A LARGE SIGNALING COMPEX WITH PROTEIN TYROSINE
KINASE ACTIVITY.
4. RECRUITMENT OF OTHER SIGNAL TRANSDUCTION PATHWAYS: signals
from the BCR and TCR results in the production of the second
messenger IP3 and DAG.
IP3 RELEASE Ca2+

DAG ACTIVATE PKC

SMALL G PROTEINS RAS and Rac are activated

• Change in pattern of gene expression

• Functional changes in cells
• Differentiation and activation.
THE B CELL Co RECEPTOR COMPEX CAN ENHANCE B CELL
RESPONSES:
• Stimulation through antigen receptor can modified into signals through co –
receptor.
• Co stimulation through CD28 cause effective stimulation of T-lymphocyte while
signaling through CTLA-4 inhibits the response of T cells.
• B cells a component of B cell membrane called B cell co receptor provides
stimulatory modifying signals.
• The B cell co receptor consist of 3 proteins :
1. CD19:
• Long cytoplasmic tail
• 3 extracellular domains
2. TAPA1:(TARGET OF ANTIPROLIFERATIVE ANTIBODY 1)
• transmembrane protein
3. CR2: receptor for membrane molecule
• Receptor of C3d which has coreceptor activity
• Breakdown product of complementary system which destroy invaders.
CR2 co receptor complex binds complement
coated antigen that has been captured
by mIg of B cell.

This crosslinks the co receptor to the BCR

and allow CD19.

CD19 interact with Igα/Igβ component of BCR

• (6 Tyrosine residues in cytoplasmic tail)
• (protein tyrosine kinase activate by
Crosslinking of BCR)

Phosphorylation leads to bind number of signaling molecule.

MARKERS ON B CELLS:
MARKERS FUNCTION

CD40 • Help in form T-B conjugate( with T cell of CD40L)

• Belongs to the tumor necrosis family
• Regulate cell proliferation and apoptosis.

B7 B7-1 and B7-2 are molecules interact with CD28 and

CTLA-4 (on T CELLS)

FcyR II(CD32) Receptor for IgG

CR1(CD35) AND CR2(CD21) Receptor for certain complement products.

CLASS II MHC Permits the B cell to function as an antigen presenting

cell(APC)

B220(FORM OF CD45) Marker for B cell and their precursors.

REFERNCES:
• IMMUNLOGY – JANIS KUBY
• ESSENTIALS OF IMMUNOLOGY : SK GUPTA
• www.biologydicussion.com
Thankyou…